The double helical structure of DNA mandates the existence of a mechanism for unwinding the helix to expose ssDNA for use as a template or reaction intermediate in DNA replication, repair, and recombination. The DNA helicases provide such a mechanism by unwinding duplex DNA using energy provided by NTP hydrolysis. More than ten helicases have been described in the bacterium E. coli; the role of each in DNA metabolism is currently being elucidated. The long-range goal of this research program is to understand, in enzymatic and molecular terms, the mechanism of action and cellular role of E. coli DNA helicases Il, IV and Rep protein. The first two specific aims of this proposal involve structure-function studies of DNA helicase Il, the uvrD gene product. Highly conserved amino acid residues (within the class of helicase enzymes) will be altered by site- specific mutagenesis, and the mutant protein product will be analyzed biochemically and genetically. This approach will provide new data regarding the reaction mechanism, and is expected to yield new mutants to test the role of both ATPase and helicase activity in excision repair, methyl-directed mismatch repair, and DNA replication. In addition, a semi- random mutagenesis procedure will be used as a tool to begin to define the various activity domains likely to be present in helicase II. The third specific aim involves genetic and biochemical studies designed to elucidate the role of helicase IV in the cell. Efforts will focus on a role for helicase IV in recombination. The fourth specific aim centers on the possibility that Rep protein utilizes its helicase activity to displace proteins bound on DNA. This activity may be important in DNA replication since the replication fork must encounter protein-DNA complexes during the course of DNA synthesis. In addition, these studies begin to address the larger issue of how helicases deal with protein-bound DNA during the course of an unwinding reaction.